Purification Of Thrombin Research Articles

Molecular imprinted based microcryogels for thrombin purification

In addition to understanding and explaining the functions of proteins, the need for low-cost, easy and efficient purification methods has been increasing in the field of protein purification, which is also important for enzyme production. In this context, an alternative approach has been developed for the purification of thrombin, which has a crucial role in the hemostatic process, via thrombin imprinted microcryogels that allow reuse and have high selectivity. The characterization studies of the microcryogels were accomplished with micro-computed tomography (µCT), scanning electron microscopy (SEM), optical microscope, surface area measurements (BET analyses) and swelling test measurements. By scanning various parameters affecting thrombin adsorption, the maximum thrombin adsorption capacity (Qmax) was found to be 55.86 mg/g. Also, the selectivity of microcryogels was investigated with the competitive agents and reusability studies were performed. The purity of thrombin was evaluated by Fast Performance Liquid Chromatography (FPLC) method. Experimental results indicated that adsorption of thrombin by the developed microcryogels fit the Langmuir isotherm model (Qmax: 55.86 mg/g, R2: 0.9505) and pseudo-second order for three different thrombin concentrations (R2: 0.9978, R2: 0.9998, R2: 0.9999).

Reconstruction of the Native Biosynthetic System of Carotenoids in E. coli─Biosynthesis of a Series of Carotenoids Specific to Paprika Fruit

Capsanthin, capsorubin, cucurbitaxanthin A, and capsanthin 3,6-epoxide, a series of carotenoids specific to the red fruit of paprika (Capsicum annuum), were produced in pathway-engineered Escherichia coli cells. These cells functionally expressed multiple genes for eight carotenogenic enzymes, two of which, paprika capsanthin/capsorubin synthase (CaCCS) and zeaxanthin epoxidase (CaZEP), were designed to be located adjacently. The biosynthesis of these carotenoids, except for capsanthin, was the first successful attempt in E. coli. In a previous study, the levels of capsanthin synthesized were low despite the high expression of the CaCCS gene, which may have been due to the dual activity of CaCCS as a lycopene β-cyclase and CCS. An enhanced interaction between CaCCS and CaZEP that supplies antheraxanthin and violaxanthin, substrates for CaCCS, was considered to be crucial for an efficient reaction. To achieve this, we adapted S·tag and S-protein binding. The S·tag Thrombin Purification Kit (Novagen) is merchandized for in vitro affinity purification, and S·tag-fused proteins in the E. coli lysate are specifically trapped by S-proteins fixed on the agarose carrier. Furthermore, S-proteins have been reported to oligomerize via C-terminal swapping. In the present study, CaCCS and CaZEP were individually fused to the S·tag and designed to interact on oligomerized S-protein scaffolds in E. coli, which led to the biosynthesis of not only capsanthin and capsorubin but also cucurbitaxanthin A and capsanthin 3,6-epoxide. The latter reaction by CaCCS was assigned for the first time. This approach reinforces the scaffold's importance for multienzyme pathways when native biosynthetic systems are reconstructed in microorganisms.

Preparation of magnetic poly(ethylene glycol dimethacrylate-N-Methacryloyl-(L)-glutamic acid) particles for thrombin purification

In this study, magnetic poly(ethylene glycol dimethacrylate-N-methacryloyl-(L)-glutamic acid) (mPEGDMA-MAGA) particles were prepared by the dispersion polymerization in order to purify thrombin effectively. mPEGDMA-MAGA particles were synthesized by adding different ratios of magnetite (Fe3O4) to the medium in addition to the monomer phases EGDMA and MAGA. The characterization studies of mPEGDMA-MAGA particles were used by fourier transform infrared spectroscopy, zeta size measurement, scanning electron microscopy and electron spin resonance. mPEGDMA-MAGA particles were used in thrombin adsorption studies from aqueous thrombin solutions in both batch and magnetically stabilized fluidized bed (MSFB) system. Maximum adsorption capacity in pH 7.4 phosphate buffer solution is 964 IU/g polymer and 134 IU/g polymer in MSFB system and batch system, respectively. The developed magnetic affinity particles enabled the separation of thrombin from different patient serum samples in one step. It has also been observed that magnetic particles can be used repeatedly without significant reduction in adsorption capacity.

Heparin-doped affinity electromembranes for thrombin purification

Heparin-doped affinity electromembrane was fabricated by synthesizing conducting polymers polypyrrole onto porous polyethersulfone membrane using heparin as both ligand and dopant. The obtained affinity electromembrane exhibited a uniform conductivity value of 1.73 × 10 −4 S/cm. The content of heparin of affinity electromembrane was ∼0.894 mg/g membrane. Such heparin-doped affinity electromembranes were used to purify thrombin. The adsorption mechanism of thrombin and the effects of temperature, initial concentration of thrombin and potential on adsorption were investigated by batch experiments. The results showed that the adsorption capacity increased with increasing the temperature, initial concentration of thrombin and potential, and the adsorption isotherm fitted the Freundlich model well. Elution of protein showed desorption ratio was up to 93.1% using 2.0 M NaSCN solution as the desorption agent. The adsorption capacities of all the tested affinity electromembranes did not significantly change during the repeated adsorption–desorption operations.

The Study of Purifying Plasma Thrombin Using Chitosan Affinity Magnetic Microspheres

Nanometer Fe3O4 particle was synthesized by coprecipitation, magnetic polymeric microspheres- magnetic chitosan microspheres, containing a magnetic core and a polymer shell, were prepared by the suspension cross-linking technique. Novel affinity magnetic microspheres were obtained after immobilizing heparin onto the nanoparticles. The size, morphology, structure and magnetic response properties were studied by means of SEM, FT-IR and XRD and so on. The novel affinity magnetic microspheres′ properties for thrombin were examined by its adsorption and purification, and compared with the traditional means of column chromatography— DEAE-Sepharose Fast Flow. The results showed the affinity magnetic microspheres had a spherical appearance, narrow distribution of diameter, the particle diameter is about 50 nm, and one-step purification of thrombin using it the specific activity was 1 879.7 U/mg, recovery rate was 85% and purification fold was 11.057. Whereas, purification of thrombin by column chromatography, the product exhibits an activity of 909.84 U/mg, recovery rate of 72% and purification fold of 5.33. In conclusion, chitosan nanometer affinity magnetic microspheres was prepared and magnetic separation techniques was applied for purification of thrombin, the results is better than that from traditional methods and these results have some references values in the production and subsequent study on the research and development.

Further Removal of Factor V Related Antigen From Bovine Thrombin by Utilizing a Membrane-Filtration Step

Topical bovine thrombin is commonly used during surgery to maintain hemostasis and is rarely associated with abnormalities in hemostasis, including coagulopathies and bleeding. Coagulopathies may be related to the formation of cross-reactive antibodies to bovine factor V. Effectiveness of a new filtration step to remove factor V/Va from bovine thrombin was evaluated. A highly sensitive and specific Western blot capable of detecting minute amounts of factor V/Va and/or its fragments was developed. Samples were evaluated for bovine factor V related antigens using the Western blot method and a competitive enzyme-linked immunosorbent assay. Factor Va light chain fragment levels were detectable in crude thrombin and chromatographically purified thrombin but not in chromatographically purified and virally filtered preparations. Therefore, inclusion of the viral-filtration step during purification of thrombin is effective in reducing factor V or its fragments to undetectable levels, thus enhancing product purity.

Argatroban‐coupled Affi‐Gel matrix for the purification of thrombin from plasma

Sometimes it is necessary to obtain thrombin from limited amounts of human plasma for laboratory assay. None of the available purification methods easily deals with this subject. The procedure described in the present paper uses a readily available pharmaceutical agent, argatroban, to construct an affinity matrix. Argatroban has a high affinity for thrombin and its thrombin binding is reversible. Prothrombin derived from a Ba(2+) precipitate of human plasma is used as the starting material. The crude prothrombin can be bulk activated to thrombin using taipan-snake (Oxyuranus scutellatus) venom and bound to the argatroban-coupled matrix without further processing steps. The thrombin product eluted from the argatroban matrix is very pure as judged by high specific activity and by electrophoresis. This purification scheme is rapid, yielding purified thrombin within 2 days.

Activation-dependent Conformational Changes in β-Arrestin 2

Beta-arrestins are multifunctional adaptor proteins, which mediate desensitization, endocytosis, and alternate signaling pathways of seven membrane-spanning receptors (7MSRs). Crystal structures of the basal inactive state of visual arrestin (arrestin 1) and beta-arrestin 1 (arrestin 2) have been resolved. However, little is known about the conformational changes that occur in beta-arrestins upon binding to the activated phosphorylated receptor. Here we characterize the conformational changes in beta-arrestin 2 (arrestin 3) by comparing the limited tryptic proteolysis patterns and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) profiles of beta-arrestin 2 in the presence of a phosphopeptide (V(2)R-pp) derived from the C terminus of the vasopressin type II receptor (V(2)R) or the corresponding nonphosphopeptide (V(2)R-np). V(2)R-pp binds to beta-arrestin 2 specifically, whereas V(2)R-np does not. Activation of beta-arrestin 2 upon V(2)R-pp binding involves the release of its C terminus, as indicated by exposure of a previously inaccessible cleavage site, one of the polar core residues Arg(394), and rearrangement of its N terminus, as indicated by the shielding of a previously accessible cleavage site, residue Arg(8). Interestingly, binding of the polyanion heparin also leads to release of the C terminus of beta-arrestin 2; however, heparin and V(2)R-pp have different binding site(s) and/or induce different conformational changes in beta-arrestin 2. Release of the C terminus from the rest of beta-arrestin 2 has functional consequences in that it increases the accessibility of a clathrin binding site (previously demonstrated to lie between residues 371 and 379) thereby enhancing clathrin binding to beta-arrestin 2 by 10-fold. Thus, the V(2)R-pp can activate beta-arrestin 2 in vitro, most likely mimicking the effects of an activated phosphorylated 7MSR. These results provide the first direct evidence of conformational changes associated with the transition of beta-arrestin 2 from its basal inactive conformation to its biologically active conformation and establish a system in which receptor-beta-arrestin interactions can be modeled in vitro.

Polypyrrole–heparin system for the separation of thrombin

The development of a novel method for the purification of thrombin based on specific interaction with heparin, incorporated into polypyrrole (PPy) during growth, is described. Both platinised polyvinylidene fluoride (PVDF) membrane and reticulated vitreous carbon (RVC) were used as host substrates for the electrochemical deposition of polypyrrole–heparin. The heparin used was either unfractionated (Hep) or low-molecular-weight fractions with molecular weights of 3000 (Hep 3000) or 6000 (Hep 6000). A toluidine blue assay showed that using unfractionated heparin, a greater degree of excess sulfate groups existed in the biocomposite. However, thrombin binding was greatest for PPy–Hep 3000 and least for PPy–Hep. The use of applied potential (−0.3 V vs. Ag/AgCl (3 M NaCl)) resulted in an increase in thrombin binding to the polymer, but reversing the potential failed to release the bound thrombin. Nevertheless, thrombin release was achieved by use of appropriate buffer or 4 M NaCl solutions.

Expression, Purification, and in Vitro Characterization of the Human Outer Mitochondrial Membrane Receptor Human Translocase of the Outer Mitochondrial Membrane 20

In order to investigate the biochemical properties of the mitochondrial outer membrane receptor, hTom20, involved in protein recognition, the cytosolic domain of this receptor was overexpressed and purified to homogeneity. A four-step purification including the purification of thrombin is described as well as an analysis of the function of the highly purified hTom20 protein. The receptor was concentrated and the subsequent aggregation behavior was investigated in order to understand the function of the single cysteine in the cytosolic domain as well as the function of the proposed “glutamine face” for the structure of the protein. It was found that specific dimerization of the cytosolic domain of hTom20 is necessary in order to prevent aggregation of the protein. In addition, the cysteine and the glutamine face are important for the stability of the protein. We propose that the function of the cysteine is to promote dimerization as found in the absence of dithiothreitol.

Immobilized Hirudin and Hirudin-Based Peptides Used for the Purification of Recombinant Human Thrombin Prepared from Recombinant Human Prothrombin

A simple and efficient activation-affinity purification system was developed to obtain thrombin from recombinant CHO cells expressing human prothrombin. In this method, a controllable process for the activation of recombinant prothrombin is directly coupled with a purification strategy for the recombinant thrombin generated. At a constant flow rate and with a contact time limited to few seconds, recombinant prothrombin was filtered through immobilized trypsin. In a closed flow system, the recombinant thrombin generated was filtered through newly designed thrombin-specific affinity gels. Hirudin, the most specific thrombin inhibitor, and hirudin-based peptides were covalently immobilized to Sepharose, thus creating thrombin-specific affinity gels that immediately absorb the thrombin generated from the activation mixture. Prothrombin and incompletely activated molecules did not bind to the affinity gel and were recirculated for a further activation cycle. Due to the specificity of the affinity gels for thrombin and the elimination of thrombin from the activation mixture, proteolytic degradation and autocatalytic inactivation of the recombinant thrombin was prevented. Recombinant thrombin was isolated from the hirudin-based affinity gels by chaotrope salt elution, resulting in high yields of highly pure, active thrombin. Affinity purification of thrombin was not deleteriously affected by contamination of the starting material with other proteins. Activation and affinity purification were equally effective for recombinant and human plasma-derived prothrombin as well as for human and recombinant thrombin.

Characterisation of silica-based heparin-affinity adsorbents through column chromatography of plasma fractions containing thrombin

Different heparin-silicas, synthesised in this laboratory via directional end-point attachment of heparin (H) onto various amino-derivatised silicas, have been evaluated in packed-bed and expanded-bed column chromatographic experiments using crude preparations of the therapeutic protein, thrombin (T). Adsorbent capacities, determined through batch adsorption experiments, were verified by employing frontal analysis in packed-bed systems. The performance of these adsorbents was also investigated in terms of thrombin purification factors and recoveries. The potential of the heparin-silicas was further examined in the expanded-bed column chromatographic mode using a scaled-up procedure. With heparin-Fractosil 1000 adsorbents, capacities of around 100 000 U thrombin/ml adsorbent could be achieved. Heparin-Fractosil 1000 adsorbents of intermediate heparin content (around 4 mg heparin/ml sorbent) displayed binding stoichiometries similar to that of the commercial heparin-Sepharose (2.6–2.7 mol T/mol H). Furthermore, binding stoichiometries were largely unaffected by increasing the heparin content on the heparin-Fractosil 1000 adsorbents from 0.8 to 4.6 mg of heparin/ml of sorbent. This result suggests that optimal binding site accessibilities for the thrombin-heparin interaction occurs at lower ligand density values. The binding capacity values determined from frontal analysis were confirmed by the recovery data, thus indicating minimal irreversible adsorption. Specific activities of ca. 2100 U/mg were obtained for thrombin when affinity-purified on these heparin-LiChroprep Si60 or heparin-Fractosil 1000 adsorbents. These values were higher than the maximum achievable purity obtained through alternative, multi-step chromatographic purification procedures reported by other investigators. These results indicated that the packed-bed performances with these silica-based adsorbents were superior to currently available commercial soft gel adsorbents, with the more dense heparin-silicas exhibiting very good potential for use in expanded-bed applications.

Purification of plasma protein.

The purification of coagulation proteins from plasma milieu is a complex and sometimes difficult task. Most clotting factors are present in plasma in trace amounts. They are sensitive to proteolytic degradation and adsorption to surfaces. Thus for most coagulation proteins it was not until the seventies that highly purified preparations were available. Nowadays, most coagulation proteins are isolated from plasma using combinations of ion exchange, molecular sieve and affinity chromatographies. Sometimes, e.g. for Vitamin K dependent factors, specific adsorption properties are utilized. During affinity chromatography, proteins may be bound to specific immobilized receptors, antibodies, substrates, or inhibitors. An example for the latter is the use of bezamidine-Sepharose for the purification of thrombin. Immunoadsorption to immobilized monoclonal antibodies allowed the isolation of highly purified factor VIII:C. For factor XI and prekallikrein the binding of these proteins to the immobilized cofactor high molecular weight kininogen is utilized for an effective separation of contaminations. Antithrombin III can be isolated by linkage to its cofactor, heparin, on heparin-Sepharose. Using affinity chromatography on lysine-Sepharose it is possible to isolate plasminogen from plasma, serum or urine in a one step procedure with high yield and quality. This, of course, is an ideal situation, where the goal of a purification, namely the separation of a highly purified, biologically active product with high yield can be accomplished in a simple, fast procedure.

Thrombin purification by one-step preparative affinity chromatography on modified polystyrenes

Insoluble polystyrenes substituted with sulphonate and l-arginyl methyl ester have been synthesized. Using their specific affinity for thrombin, we developed a simple one-step chromatographic procedure for thrombin purification. As a control, insoluble polystyrenes substituted only with sulphonate groups were tested. The results obtained confirmed the importance of the arginyl residues grafted onto these polymers to obtain an affinity matrix useful for purifying thrombin with a high specific activity and a good recovery.

Purification of thrombin by affinity chromatography on immobilized heparin

Bovine thrombin has been purified from commercial crude thrombin preparations by affinity chromatography on immobilized heparin. This procedure was shown to be superior to affinity chromatography on matrix-linked synthetic low-molecular-weight thrombin inhibitors in that the purified enzyme could be conveniently eluted by a salt gradient and also was of higher purity. The degree of purification of the affinity chromatography step was 30 times and the purified material had a specific activity of about 2000 NIH units/mg. Dodecyl sulphate-polyacrylamide gel electrophoresis indicated the presence of the native two-chain form of thrombin as well as of several forms of thrombin with specific proteolytic cleavages in the B-chain. These modified forms were similar to those also found in thrombin preparations obtained from crude commercial thrombin by other methods. Affinity chromatography on heparin-agarose was shown to be useful also for the purification of thrombin from a prothrombin activation mixture.

Synthesis of heparin-sepharoses and their binding with thrombin and antithrombin-heparin cofactor

Heparin was linked covalently to Sepharose by a reaction between a uronic acid-carboxyl group of heparin and aminohexyl-Sepharose. The characteristics of this material in binding thrombin and antithrombin-heparin cofactor, was compared with those of heparin-aminoethylbromide method (heparin-Sepharose I). Although all the preparations bind thrombin and antithrombin-heparin cofactor they differ in their binding properties and purification efficiency. Highly purified antithrombin heparin cofactor was isolated by adsorption on heparinaminohexylsepharose and desorption with 1.0 M NaCl. The greatest purification of thrombin is obtained by absorption on heparin-Sepharose I and successive elution with 0.3 M and 0.4 M NaCl. Heparin-Sepharose prepared from partially degraded heparin has a relatively low affinity for thrombin and antithrombin-heparin cofactor.

Affinity chromatograpy: Purification of bovine trypsin and thrombin

Bovine trypsin has been purified by affinity chromatography on agarose beads containing covalently bound p-aminophenylguanidine, p-aminobenzamidine, or m-aminobenzamidine. Bovine thrombin was purified on a m-aminobenzamidine-agarose column containing a high concentration of the inhibitor. The values of the inhibition constant, K i, for these inhibitors were determined for both enzymes and found to be 5–10 times poorer for thrombin than for trypsin. Only those benzamidines with low K i values and coupled in high concentration to the agarose matrix were satisfactory for thrombin purification. Affinity-purified trypsin and thrombin were both greater than 90% active as measured by active site titration.

The Purification of Thrombin and Isolation of a Peptide Containing the Active Center Histidine

A chromatographic purification of bovine thrombin from commercial starting material is described which yields preparations judged to be essentially pure, that is, with a clotting activity of 2100 to 2500 NIH units per mg. In addition to the two-chain form of thrombin, the purified enzyme contains a three-chain form arising from cleavage of the B chain at arginine-73 (or 76). This form must be fully active since the specific clotting activity of various preparations does not vary with the content of the three-chain form, amounting at times to 70%. Inactivation of thrombin with Nα-tosyl lysyl chloromethyl ketone and Nα-(p-nitrobenzyloxycarbonyl)arginyl chloromethyl ketone results in alkylation of nitrogen-3 of the active center histidine with loss of both clotting and esterase activities. A radioactive peptide has been isolated from thrombin inactivated with tritiated Nα-tosyl lysyl chloromethyl ketone and shown to contain histidine-43.

Bovine Thrombin and Activated Factor X: SEPARATION AND PURIFICATION

Abstract Parke-Davis bovine thrombin was resolved into three distinct protein peaks on a Sephadex G-200 column. A stepwise chromatographic technique on diethylaminoethyl (DEAE) cellulose was developed for the complete separation and partial purification of thrombin and activated Factor X. Thrombin was quantitatively eluted in the first protein peak with 0.1 m NaCl, pH 7.0. A second peak with residual thrombin activity, containing more than 65% of the applied protein, was eluted with 0.14 m NaCl in 0.05 m sodium citrate, pH 5.8. When this fraction was rechromatographed, to remove the residual thrombin, Factors II, VII, IX, and nonactivated X were detected in this peak. Activated Factor X was eluted from the column with 0.2 m sodium citrate, pH 5.8. This activated Factor X fraction did not clot a standard fibrinogen solution during 24 hours either at 22° or 37°, with or without added calcium, nor after removal of the citrate by dialysis against 0.14 m NaCl. The DEAE-cellulose-chromatographed thrombin was further purified by Sephadex G-200 chromatography. More than 68% of the resulting thrombin peak had a constant specific activity of approximately 7000 National Institutes of Health units per mg of tyrosine. At high dilutions, when the thrombin activity in the commercial bovine thrombin preparation was negligible, the activated Factor X activity was still detectable in appreciable quantity. The purified thrombin fraction was free of plasminogen and plasmin.

Purification of thrombin by chromatography

Purification of thrombin by chromatography